Extracts and compounds from &#34;agapanthus africanus&#34; and their use as biological plant protecting agents

ABSTRACT

The invention relates to plant extracts, especially based on species of the genus  Agapanthus  and combinations thereof with other extracts deriving from other plants. The invention further relates to the isolation, purification and identification of compounds in these extracts. The plant extracts and the isolated substances show significant antimicrobial activity, especially antifungal activity, and bio-stimulatory efficacy, when applied to other plants in vitro and in vivo, including under field conditions. The products according to this invention are suitable to be used as plant protecting agents for many crops and economic plants as an alternative for chemical pesticides.

TECHNICAL FIELD OF THE INVENTION

The invention relates to plant extracts, especially based on species ofthe genus Agapanthus and combinations thereof with other extractsderiving from other plants. The invention further relates to theisolation, purification and identification of compounds in theseextracts. The plant extracts and the isolated substances showsignificant antimicrobial activity, especially antifungal activity, andbio-stimulatory efficacy, when applied to other plants in vitro and invivo, including under field conditions. The products according to thisinvention are suitable to be used as plant protecting agents for manycrops and economic plants as an alternative for chemical pesticides.

BACKGROUND OF THE INVENTION

Worldwide agriculture suffers, especially in developing countries suchas in Africa, from annual huge losses of crop and other economic plantsdue to plant diseases. More than 30% of the food, fiber, feed and energyproduced in crop production systems are destroyed by insects anddiseases annually on a global scale. These yield losses are high as aresult of low-input production systems due to the non-affordability ofsynthetic fungicides to farmers in developing countries that depend onnon-conventional disease management practices often providing doubtfulresults.

In contrast, crop and plant producers in developed countries relylargely on synthetic pesticides to control plant diseases. It is anestablished fact that the use of synthetic chemical pesticides providesmany benefits to crop producers. These benefits include higher cropyields, improved crop quality and increased food production for an everincreasing world population. The development of a wide range ofchemicals with different formulations has enabled man to control a widerange of plant pathogens and substantially increased crop yields. Morethan a decade ago crop producers spent nearly $20 billion on pesticidesand $150 million on other plant protection techniques, worldwide, tocontrol pests in general. The world market share of fungicides alone was20% in recent years whilst Europe accounted for 30% of the market.However, the same level of pathogen control has not been realized indeveloping countries, partly as a result of pesticide technology notbeing accessible to most resource poor farmers. Failure of modernapproaches, technology and chemicals to reach farmers in developingcountries is solely the result of high costs in relation to the value ofthe crops cultivated by these farmers. Consequently, crops are routinelysubjected to attack from a wide spectrum of a diversity of pathogens andthese farmers constantly experience serious crop damage. Moreover, yieldlosses are on the increase despite high pesticide usage, even indeveloped countries. Furthermore, control of plant diseases is noteasily achieved with a single application of fungicide but requiresfrequent applications during the crop-growing period. However, syntheticpesticides may pose a couple of threats and hazards to the environment,especially when improperly used by farmers in developing countries wholack the technical skill of handling them, and who fail to adopt to thistechnology easily. This may result in undesirable residues left in food,water and the environment, and may cause toxicity to humans and animals,contamination of soils and groundwater and may lead to the developmentof crop pest populations that are resistant to treatment withagrochemicals. Especially sulfur and copper containing syntheticfungicides are toxic to mammals, wildlife and many beneficial insects.

Furthermore, in Africa and the Near East, obsolete pesticides havebecome a source of an additional great environmental concern. Somestocks are over 30 years old and are kept in poor conditions because ofinadequate storage facilities and lack of staff trained in storagemanagement. Obsolete pesticide stocks are potential time bombs. Leakage,seepage and various accidents related to pesticides are quite common andwidespread. Additionally, that frequent application of fungicides hasresulted in fungal mutation and, subsequently new resistant strains(Khun, 1989, Pesticide Science 14:272-293), the combat of which usuallyrequires stronger pesticides with again stronger impacts on theenvironment.

For all these reasons there is a considerable and increasing consumerresistance especially in the developed countries, initiated politicallyby the green parties, towards the use of synthetic chemicals/pesticidesespecially, supplying a rationale for a shift from chemical pesticidesapplications to the use of naturally derived plant protecting agents inorder to reduce the pollution and health risk caused by pesticides.

As a result, research on the possible utilization of biologicalresources and its application potential in agriculture has become veryrelevant. A promising approach in this regard is the use of naturalplant products as an interesting alternative to synthetic chemicals dueto the apparent less negative impact on the environment.

This especially applies to the search for environmentally friendlybioactive naturally derived components and agents with, for example,broad-spectrum antimicrobial activity.

Natural products from plants are expected to have a narrow target rangeand highly-specific mode of action, to show limited field persistence,to have a shorter shelf life and present no residual threats. They aregenerally safer to humans and the environment than conventionalsynthetic chemical pesticides and can easily be adopted by farmers indeveloping countries who traditionally use plant extracts for thetreatment of human diseases.

A further rationale for exploring the use of plant extracts or naturalproducts as biological pesticides more extensively can be found in theplant itself. Plants have evolved highly specific chemical compoundsthat provide defense mechanisms against attack by disease causingorganisms, including fungal attack, microbial invasion and viralinfection (Cowan, 1999, Clinical Microbiology Reviews 12:564-582). Thesebioactive substances occur in plants as secondary metabolites, and haveprovided a rich source of biologically active compounds that may be usedas novel crop-protecting agents. In nature some plants have thepotential to survive very harsh environmental conditions. This hasinitiated the postulate that such plants might be utilized as sourcesfor the development of natural products to be applied in agriculture byman as natural herbicides, bactericides, fungicides or products in crudeor semi-purified form. Secondary plant metabolites are distinct fromprimary metabolites in that they are generally non-essential for thebasic metabolic processes such as respiration and photosynthesis. Theyare numerous and widespread, especially in higher plants and oftenpresent in small quantities (1-5%) as compared to primary metabolites(carbohydrates, proteins, lipids). Secondary metabolites are probablyproduced when required in the plant system and are synthesized inspecialized cell types. Ecologically, secondary metabolites playessential roles in attracting pollinators, as adaptations toenvironmental stresses and serve as chemical defenses against insectsand higher predators, micro-organisms and even other plants(allelochemicals).

Abiotic stress such as nutrient limitation, light intensity, waterstress and others has been considered to trigger the formation ofsecondary metabolites. A biotic stress related type of plant-pathogeninteraction involves the production of metabolites as part of a plantdefense arsenal against microbial invasion and is considered diseasedeterminants. Secondary metabolites with anti-microbial propertiesinclude terpenoids (e.g. iridoids, sesquiterpenoids, saponins),nitrogen- and/or sulphur containing (e.g. alkaloids, amines, amides),aliphatics (especially long-chain alkanes and fatty acids) and aromatics(e.g. phenolics, flavonoids, bi-benzyls, xanthones and benzoquinones).

Another related area of organic farming systems is the potential toapply natural plant extracts as either plant growth regulators orbio-stimulants. Many natural plant compounds have been identified thataffect the growth and development of plants. Secondary metabolites fromplants may show also bio-stimulatory activities in plants, other plantsincluded. Probably the most effective compound to enhance crop yield,crop efficiency and seed vigour has been identified as a brassinosteroid(Mandava, 1988, Plant Physiology Plant Molecular Biology 39:23-52).Brassionosteroids have also been identified as bio-stimulatorysubstances from a plant extract mixture deriving from a specific Pinkspecies and a specific Alfalfa species (EP 1 051 075 B1). An elevatedinterest therefore exists to identify natural plant compounds with theability to manipulate plant growth and development over a short period,e.g. a growing season. An additional consideration is that plants whoseextracts, for example show antimicrobial and/or bio-stimulatoryproperties, could be cultivated as alternative agricultural crops forserving as sources of active compounds in the production of naturalpesticides or plant growth regulators.

Although plants are a valuable source for the development of new naturalproducts with the potential to be used for disease management in organiccrop production systems only a small number of plants has beeninvestigated for possible use in plant disease control in agriculture.However, related to this relatively small number of investigated plantsa relatively large number of scientific research activities has beendone during the last couple of years. Some of them are listed asfollows:

-   -   It was shown (Pretorius et al., 2002, Annals of Applied Biology        141:117-124) that mycelial growth inhibition was obtained with        extracts from two species of the subclass Liliidae, namely        Aristea ecklonii and Agapanthus inapertus. The crude extract        of A. ecklonii performed best of all extracts as it totally        inhibited the mycelial growth of all seven of the plant        pathogenic test organisms and outperformed the inhibition by a        broad spectrum synthetic fungicide (carbendazim/difenoconazole).        Crude extracts of A. inapertus showed complete inhibition of        four and strong inhibition of the remaining three plant        pathogenic fungi.    -   Plant seeds also contain compounds with antimicrobial        properties. Seed extracts of 50 plant species, belonging to        different families, were evaluated for their ability to inhibit        the growth of Trichoderma viride in vitro (Bharathimatha et al.,        2002, Acta Phytopathologica et Entomologica Hungarica 37:75-82).        Of the various seed extracts, that of Harpullia cupanioides        (Roxb.), belonging to the family Sapindaceae, displayed very        high antifungal activity.    -   The natural plant product Milsana®, extracted from the giant        knotweed (Reynoutria sacchalinensis), is probably best known        (Daayf, 1995, Plant Disease 79:577-580). The product has been        reported to control powdery mildew, caused by Sphaerotheca        fuliginea, in long English cucumber under greenhouse conditions        and also showed broad spectrum activity against powdery mildew        of tomato, apple and begonia as well as downy mildew of        grapevine and rust of bean.    -   Extracts from the leaves and seed kernels of the neem tree (Ume        et al., 2001, The science and application of neem, meeting        proceedings, Glasgow, U.K. April-2001. Pp. 33-37) were tested        for antifungal activity against the plant pathogenic fungus        Sclerotium rolfsii [Corticium rolfsii]. All the extracts showed        some effect against different growth stages of the fungus, but        the effects were fungistatic rather than fungitoxic.    -   Amadioha (2002, Archives of Phytopathology and Plant Protection        35:37-42) evaluated the antifungal activities of the different        extracts of A. indica. The oil extract from seeds as well as        water and ethanol leaf extracts of the plant were effective in        reducing the radial growth of Cochliobolus miyabeanus in culture        and in controlling the spread of brown spot disease in rice.    -   A study directed towards identifying bio-stimulatory properties        in plant extracts was performed by Cruz et al. (2002, Acta        Horticulturae 569:235-238) by treating the roots of bean, maize        and tomato with an aqueous leachate of Callicarpa acuminate. The        aqueous extract of C. acuminata inhibited the radical growth of        tomato but had no effect on root growth of maize or beans.    -   According to Singh et al. (2001, Journal of Crop Production        4:121), allelochemicals isolated from some plants show strong        bio-herbicidal activity at high concentrations, but at low        concentrations these extracts can promote crop seed germination        and seedling growth, hence showing a potential to be applied as        bio-stimulatory agents or growth promoting substances in        agriculture.    -   Extracts from some lucerne cultivars had a stimulatory effect in        terms of seed germination as well as root and hypocotyl growth,        whereas others showed the direct opposite effect, confirming        that crop plants can also be affected by plant extracts aimed at        controlling weed growth (Tran and Tsuzuki, 2002 Journal of        Agronomy and Crop Science 188:2-7).    -   Leksomboon et al. (2001, Kasetsart Journal, Natural Sciences        35:392-396) demonstrated the antibacterial effect of leaf and        other aqueous extracts of Hibiscus sabdariffa, Psidium guajava,        Punica granatum, Spondias pinnata and Tamarindus indica against        Xanthomonas axonopodis, the casual agent of citrus canker under        both laboratory and field conditions.    -   Another natural product, carvone, derived from dill and caraway        seed, has been developed to inhibit the growth of storage        pathogens and to suppress sprouting of potatoes in the warehouse        (Moezelaar et al., 1999, In: Modern fungicides and antifungal        compounds II, Intercept Limited, p. 453-467). Carvone is        currently marketed as Talent® in the Netherlands.    -   In European patent EP 1 051 075 a preparation of a combination        of species of the Pink family and species of Alfalfa is        described (ComCat®) which reveals within a specific ratio a        synergistic bio-stimulatory effect. ComCat® has demonstrated        consistent plant growth enhancement and physiological efficiency        in the treated plant's utilization of available nutrients.        ComCat®, which enhances the health of vegetables, flowers and        agricultural crops, is not a fertilizer substitute but, instead,        it is a biological enhancer which stimulates the plant to more        properly utilize available nutrients. Moreover, it activates and        induces allelopathy and disease resistance in the treated plant        and stimulates greater production of sugars, which are the        building blocks for cellulose and fruiting bodies. The result is        a more productive, healthier plant with stronger plant stalks,        better flowering and greater fruit biomass (Agraforum: Germany,        2002, Technical data sheet).

SUMMARY OF THE INVENTION

The invention provides extracts and preparations based on species of thegenus Agapanthus, preferably Agapanthus africanus, which elicit asignificant antimicrobial, preferably antifungal activity in vitro andin vivo, even under field and glasshouse conditions. Moreover, theseextracts elicit a significant bio-stimulatory activity, expressed, aboveall, by an increased growth metabolism. Extracts or preparations fromthe aerial parts of the plants show a higher efficacy as compared to thesoil parts of the plant. Furthermore, extracts or preparations from thecombined aerial parts of the plants (flowers, leaves, stalks) show ahigher antifungal and bio-stimulatory efficacy as compared to the sum ofextracts or preparations from the single components of the aerial parts,indicating that synergism is participated in the involved biologicalprocesses. Furthermore, combined extracts or preparations from speciesof the genus Agapanthus and the species Tulbaghia violacea show a higherantifungal and bio-stimulatory efficacy as compared to the extracts orpreparations of the single species and let assume the existence of asynergistic process.

The invention provides, in addition, compositions of combinations ofextracts or preparations of different plant species. These combinationscomprise-preparations from species of the genus Agapanthus, preferablyA. africanus, and other plant species, preferably garlic species, mostpreferably from Tulbaghia violacea (wild garlic). Alternatively,according to the invention, a preparation from species of the genusAgapanthus is combined with a preparation of a mixture of species of thePink family and Alfalfa species, preferably in a specific ratio. Inanother embodiment of the invention provides combinations of species ofthe genus Agapanthus with Tulbaghia violacea and a mixture of species ofthe Pink family and Alfalfa species. These combinations elicit anincreased and synergistic plant protective activity, preferably anantifungal and bio-stimulatory activity, as compared to thecorresponding single-component preparations.

The invention provides finally at least four compounds isolated andpurified from said extracts/preparations, which also show significantplant protecting activity, especially antifungal activity, when appliedto other plants in vitro and in vivo, field cultivation included. Thesefour compounds are:3-[{O-β-D-glucopyranosyl-(1″-3′)-α-L-rhamnosyl-(1″-2′)}-β-D-glucopyranosyloxy]agapanthegenin,5,7,4′ tri-O-flavanone, 5,7,3′,4′-tetra-O-acetylflavanone andtrans-4,2′,4′-tri-O-acetylchalcone.

The preparations according to the invention can be provided as crudeextracts or as dried powder dependent on the process of theirmanufacture. The preparations may comprise additionally, especially foruse in field cultivation, solid preferably pulverulent fillers orcarrier materials according to the state of the art. Moreover, thepreparations according to the invention may comprise conventionaladditives that augment or modulate the effect of the preparation.

The preparations according to the invention can be provided also in aliquid, preferably aqueous form, which can be uses as a spray, and thuscan be easily atomized on the areas under cultivation. In such solutionsor suspensions the extracts and preparations of the invention revealtheir full plant protecting activity in a concentration range between0.25 g (extract/powder)/1 to 2 g/l, preferably from 0.5 g/l to 1 g/l.With respect to the antifungal activity of the preparations, the term“full plant protecting activity” means 100% inhibition of the mycelialgrowth of a typical fungal plant pathogen compared to a standardreference pesticide.

The invention also provides processes for the manufacture of the crudeextracts and dry powder preparation based on extraction of the plants orplant parts with organic polar solvents, such as methanol or ethanol ormixtures thereof.

The invention finally provides a process of isolating, purifying andidentifying substances from said extracts which show significantantifungal and bio-stimulatory activity in diseased plants in vitro andin vivo.

In more detail the invention provides:

-   -   A preparation suitable for biological plant protection based on        plants or parts of plants from the genus Agapanthus, preferably        the species A. africanus, in form of a crude extract, whereby        said preparation is obtainable by the following process steps:        -   (i) drying the plant material preferably at 30-40° C.            preferably to the exclusion of sun light;        -   (ii) grinding the dried plant material to a grit size            between 0.2-2 mm;        -   (iii) soaking the ground material in a polar organic solvent            selected from the group consisting of methanol and ethanol,            thus forming a suspension/solution        -   (iv) performing a stirred extraction of the suspension and            separating the supernatant from the solid phase;        -   (v) repeating step (iii) and (iv) at least one additional            time; preferably two times,        -   (vi) combining the soluble organic phases of step (iv) and            removing the organic solvent by preferably vacuum            evaporation at 30-40° C., thus obtaining the crude extract            residue.    -   Alternatively, a corresponding preparation in form of a dry        powder, obtainable by the following steps:        -   (i) drying the plant material at preferably 30-40° C.,            preferably to the exclusion of sun light;        -   (ii) grinding the dried plant material to a grit size less            than 0.1 mm,        -   (iii) soaking the ground material in methanol or ethanol,            preferably methanol, thus forming a suspension/solution;        -   (iv) performing a stirred extraction of the suspension;        -   (v) evaporating the solvent without prior separation of the            solid phase from the soluble organic phase;        -   (vi) soaking the evaporated solid phase residue in ethanol            or methanol, preferably ethanol and repeating steps (iv) and            (v); and        -   (vii) drying the evaporated solid phase residue, thus            obtaining a dry powder.    -   A corresponding preparation, wherein one or more of the        different aerial parts (flowers, leaves, stalks) of the plants        are used, preferably the flowers.    -   A corresponding preparation, wherein the combined aerial parts        (flowers plus leaves plus stalks) are used; said preparation is        showing an additional (synergistic) effect as compared to the        over-all effect of the single components of the aerial parts of        Agapanthus.    -   A corresponding preparation, wherein the soil plant parts are        used.    -   A corresponding preparation comprising:        3-[{O-β-D-glucopyranosyl-(1″-3′)-α-L-rhamnosyl-(1″-2′)}-β-D-glucopyranosyloxy]agapanthegenin,        and/or 5,7,4′ tri-O-flavanone and/or        5,7,3′,4′-tetra-O-acetylflavanone and/or        trans-4,2′,4′-tri-O-acetylchalcone.    -   A corresponding preparation comprising        3-[{O-β-D-glucopyranosyl-(1″-3′)-α-L-rhamnosyl-(1″-2′)}-β-D-glucopyranosyloxy]agapanthegenin,        and 5,7,4′ tri-O-flavanone, and        5,7,3′,4′-tetra-O-acetylflavanone and        trans-4,2′,4′-tri-O-acetylchalcone.    -   A corresponding preparation, which further comprises solid,        pulverulent carrier materials or fillers and/or additives that        augment or regulate the effect of the preparation.    -   A corresponding preparation in form of an aqueous solution or        suspension, which can be easily sprayed and distributed on        fields and areas under cultivation, in which the plants to be        protected are cultivated.    -   A corresponding, wherein the concentration of crude extract or        the dry powder is in the range from 0.25 g/l to 2 g/l,        preferably from 0.5 g/l to 1 g/l.    -   A composition comprising a first plant preparation as specified        above and second plant preparation in form of a crude extract,        dry powder or an aqueous suspension or solution thereof, wherein        said second plant preparation exerts an additional or even        synergistic plant protective effect on the plants or parts        thereof treated with the composition.    -   A corresponding composition comprising as second plant        preparation a preparation deriving from garlic species,        preferably Tulbaghia violacea, whereby said second preparation        is obtained by analogous process steps as said first        preparation.    -   A corresponding composition comprising as second plant        preparation a preparation deriving from a mixture of species of        the Pink family and Alfalfa species, wherein preferably the        proportion by weight of the dried Pink species material is        between 80 and 99%, and said second plant preparation is        obtained by analogous process steps as said first plant        preparation.    -   A composition comprising (i) said first plant preparation, (ii)        said second plant preparation, and (iii) a third plant        preparation deriving from a mixture of species of the Pink        family and Alfalfa species, wherein preferably the proportion by        weight of the dried Pink species material is between 80 and 99%,        whereby each preparation is in form of a crude extract, dry        powder or an aqueous suspension or solution thereof, and said        second and third plant preparation exert an additional plant        protective effect on the plants or parts thereof treated with        the composition.    -   The use of a preparation/composition as described above as a        biological plant protective agent.    -   The use of a preparation/composition as described, wherein the        biological plant protective agent is an antimicrobial agent,        preferably an antifungal agent, which preferably inhibits or        reduces the mycelial growth of fungi and is enabled to prevent        plants, preferably crop, from infection by fungi under field        conditions.    -   The use of a preparation/composition as described above as a        bio-stimulatory agent, which exerts growth induction    -   The use of a preparation/composition as described above as a        bio-stimulatory agent, which induces systemic acquired        resistance (SAR) in plants or plant parts treated with the        agent.    -   The corresponding uses, wherein the applied Agapanthus        preparations derive from the combined aerial parts of        Agapanthus.    -   The corresponding use, wherein the activity or efficacy of said        preparation is higher than the sum of the activities or        efficacies of preparations based on the respective single        components of the aerial parts of Agapanthus.    -   A process for the preparation of a crude extract or a dry powder        preparation or aqueous suspensions or solutions thereof from        Agapanthu as defined above, comprising the following steps:        -   (i) drying plant material from Agapanthus at 30-40° C.,            preferably to the exclusion of sun light;        -   (ii) grinding the dried plant material to a grit size            between 0.1-3 mm, preferably between 0.2-2 mm;        -   (iii) soaking the ground material in a polar organic            solvent, such as methanol or ethanol, preferably 90-10%            methanol or ethanol or mixtures thereof, thus forming a            suspension/solution;        -   (iv) performing a stirred extraction of the suspension and            separating the supernatant from the solid phase;        -   (v) repeating step (iii) and (iv) at least one additional            time; preferably two times,        -   (vi) combining the soluble organic phases of step (iv) and            removing the organic solvent by preferably vacuum            evaporation at 30-40° C., thus obtaining the crude extract            residue;    -   and in the case of the preparation of an aqueous preparation;        -   (vii) suspending the resultant crude extract in water in a            suitable concentration preferably in a range between 0.1 g/1            and 2 g/l, more preferably between 0.5 g/l and 1 g/l.    -   A compound of formula I,

-   -   wherein R═H or acetyl.    -   A corresponding compound isolated from a preparation as        described, wherein said compound is        3-[{O-β3-D-glucopyranosyl-(1″-3′)-α-L-rhamnosyl-(1″-2′)}-β-D-glucopyranosyloxy]agapanthegenin.    -   A corresponding composition suitable for biological plant        protection comprising a compound of formula I or, more        specifically,        3-[{O-β-D-glucopyranosyl-(1″-3′)-α-L-rhamnosyl-(1″-2′)}-β-D-glucopyranosyloxy]agapanthegenin        and at least one flavonoid compound selected from the group        consisting of 5,7,4′ tri-O-flavanone,        5,7,3′,4′-tetra-O-acetylflavanone and        trans-4,2′,4′-tri-O-acetylchalcone.    -   The use of said isolated compounds as plant protective agent,        wherein the plant protective agent is preferably an antifungal        agent that inhibits the mycelial growth of fungi.    -   An alternative process for the preparation of a crude extract or        a dry powder preparation or aqueous suspensions or solutions        thereof derived from Agapanthus as defined above comprising the        steps        -   (ia) drying the plant material from Agapanthus at preferably            30-40° C., preferably to the exclusion of sun light;        -   (iia) grinding the dried plant material to a grit size less            than 0.1 mm,        -   (iiia) soaking the ground material in a first polar organic            solvent, preferably 90-100% methanol, using 1.0-3.0 ml/g dry            weight of the ground plant material, thus forming a            suspension/solution;        -   (iva) performing a stirred extraction of the suspension;        -   (va) evaporating the solvent without prior separation of the            solid phase from the soluble organic phase;        -   (via) soaking the evaporated solid phase residue in a second            polar organic solvent, preferably 90-10% ethanol, using            1.0-3.0 ml/g dry weight of the ground plant material, and            repeating steps (iva) and (va);        -   (viia) drying the evaporated solid phase residue, thus            obtaining a dry powder; and in the case of the preparation            of an aqueous preparation,        -   (viii) suspending/solving the resultant dry powder in water            in a suitable concentration, preferably in a range as            indicated above.

DETAILED DESCRIPTION OF THE INVENTION (A) General Definitions

Above and below terms and expressions are used which have according tothe understanding of the this invention the following meanings:

The term “plant protecting agent” or “plant protective agent” means, ifnot otherwise specified, any kind of synthetic or natural agent,product, extract, composition that is effective in a broad sense for theprotection and health of a plant against infection and damages bypathogens in vitro and/or in vivo. The term includes agents, products,extracts, compositions or single isolated components of extracts whichmay show a couple of different biological activities and/or properties,such as antimicrobial, antiviral, antifungal, and bio-stimulatoryactivity/efficacy, growth inducing/promoting activity (with respect tothe plant to be protected), growth inhibitory activity (with respect tothe plant(s) competitive to the plant to be protected), systemic and/orimmunological acquired resistance inducing/promoting activity, andallelopathy inducing/promoting activity.

The term “biological plant protection” means according to the invention,if not otherwise specified, that the protection of a plant is achievedby naturally occurring or naturally derived substances or sourcespreferably from plants, and not by synthetic or chemical means oragents, which do not occur in nature, preferably plants or part ofplants.

The term “biological plant protecting (protective) agent” is thus,consequently a plant extract, a plant preparation, a composition basedon plants or parts thereof, or an agent isolated from a plantextract/preparation/composition, which all show significant efficacyagainst a plant pathogen in vitro and/or in vivo. This term includesalso chemically synthesized compounds which are structurally andfunctionally identical with the isolated naturally derived compound, butexcludes expressively chemically synthesized pesticides and relatedcompounds having no natural derived counterpart.

The term “pesticide” means according to the invention, if not otherwisespecified, not naturally derived or occurring, synthetic compounds,agents or compositions which have plant protecting efficacy.

The term “plant pathogen” means a compound or composition or livingmaterial, such as a microorganism (including viruses), which causesdisease or damage to the plant. In a narrower scope of the invention theterm is focused to pathogenic microorganisms including metabolicproducts of these microorganisms.

The term “antimicrobial” according to the invention encompasses anefficacy or activity against microorganisms, including viruses, bacteriaand fungi, that reduces or eliminates in vitro and/or in vivo the(relative) number of active microorganisms which attack the plant orparts thereof to be protected. Thus, the term includes the terms“antiviral”, “antibacterial”, and “antifungal”. An “antimicrobial agent”according to the invention is a biological plant protecting agent asspecified above, which prevents or reduces infections or damages of aplant caused by a pathogenic microorganism.

The term “antibacterial” means according to the invention an activity orefficacy (e.g. of an agent or extract, etc.), that reduces or eliminatesthe (relative) number of active bacteria. An “antibacterial agent”according to the invention is a biological plant protecting agent asspecified above, which prevents or reduces in vitro and/or in vivoinfections or damages of a plant caused by a pathogenic bacterium.

The term “antiviral” means according to the invention an activity orefficacy (e.g. of an agent or extract, etc.), that reduces or eliminatesthe (relative) number of active viruses. An “antiviral agent” accordingto the invention is a biological plant protecting agent as specifiedabove, which prevents or reduces in vitro and/or in vivo infections ordamages of a plant caused by a pathogenic virus.

The term “antifungal” means according to the invention an activity orefficacy (e.g. of an agent or extract, etc.), that reduces or eliminatesthe (relative) number of active fungi. An “antifungal agent” accordingto the invention is a biological plant protecting agent as specifiedabove, which prevents or reduces in vitro and/or in vivo infections ordamages of a plant caused by a pathogenic fungus. The antifungalactivity may lead to the inhibition of mycelial growth as well as sporegermination of fungi.

The term “bio-stimulatory” means according to the invention, if nototherwise specified, an activity or efficacy which stimulates, increasesor improves many different processes in the plant or plant parts, suchas improved generation of growth promoting substances like sugars andamino acids, improved adequate supply of cells with available nutrientsand growth regulators, enhanced cell metabolism, improved celldecontamination, enhanced immune defense, promotion of growth and yield,induction of systemic acquired resistance (SAR), inhibition of growthand yield of competing plants (allelopathy). The bio-stimulatoryactivity can be caused by agents, plant extracts and compositionsincluding metabolic compounds synthesized by the plant to be protectedafter induction of their synthesis by said bio-stimulatory agent. A“bio-stimulatory agent” according to the invention is a biological plantprotecting agent as specified above, which shows the above-specifiedbio-stimulatory properties in a plant treated with this agent in vitroand/or in vivo.

A “plant growth regulator” is a compound or a mixture of substanceseither natural or synthetic, that modifies or controls one or morespecific physiological processes within a plant. If the compound isproduced within the plant it is called a plant hormone e.g. auxins,gibberellins, abscisic acid and ethylene.

“SAR” (Systemic Acquired Resistance) occurs in a plant or parts thereofaccording to the invention if it shows induction or enhancement ofactivity of defense or protection related enzymes (PR-proteins). Suchenzymes include, for example, peroxidase, β-1,3-glucanse and NADPHoxidase.

(B) Plant Description

Agapanthus is originally indigenous to South Africa. Studies on itsdistribution indicated that the evergreen species of Agapanthus growswildly from the south-western Cape eastwards into Natal and furtherNorth. It is also grown in Europe, America, Australia, New Zealand andSouth America.

The taxodermic classification of Agapanthus africanus is:

. Division: Magnoliophyta Class: Liliopsida Subclass: Lilidae Family:Amaryllidaceae Subfamily: Lilidaceae Genus: Agapanthus Species:Agapanthus africanus (A. umbelattus)

The genus Agapanthus (L.) Hoffmg (Alliaceae) may be divided into twogroups according to the type of flowers they bear namely those withflowers having short tubes with perianth segments spreading out widely,and those with long tubes and perianth segments that do not spread much.The genus is sometimes also divided into evergreen or deciduous types.A. africanus (synonym A. umbellatus) is the evergreen one with floweringstems of about 60 cm in length and deep blue flowers with a darkerstripe down the center of each petal. It grows 30 to 60 cm in height andhas shorter, fewer and more leathery leaves than the subspecies A.praecox (orientalis). It also has much fewer flowers, usually about 12to 18, in a smaller head than that of A. praecox and flowers fromDecember to March. There is also a rare white form, A. walshii.

A. africanus can be cultivated. It is a perennial with a large rootsystem that enables it to go without water for long periods of time. Asthe root volume increases from season to season and give rise to newplants spontaneously, roots can also be used to multiply the plant as acultivation practice. Eventually the plants begin to suffer throughbeing overcrowded. For this reason the clumps which they form should belifted every few years and divided. A. africanus grows in any kind ofsoil. To obtain good results in poor soil, it may be necessary toprepare trenches of approximately 30-45 cm deep and incorporate compostand manure. Although the plants are drought tolerant, they flower betterif watered regularly during spring and summer when flower formation isat its peak.

(C) Microorganisms

Six common South African plant fungal pathogens are chosen to test forthe fungitoxic properties of the plant extracts. These fungal pathogensincluded Botrytis cinerea Pers.:Fr. (Hyphomycetes), Fusarium oxysporumSchlechtend.:Fr. (Hyphomycetes), Sclerotinia rolltsii Sacc.(Agonomycetes), Rhizoctonia solani Kühn (Agonomycetes), Botryosphaeriadothidea (Moug.: Fr.) Ces. & De Not. (Loculoascomycetes) and Pythiumultimum Trow (Oömycetes). Plant pathogenic bacteria used in this studyinclude Agrobacterium tumefaciens Smith and Townsend, Clavibactermichiganense Spieckermann pv. michiganense Smith, Erwinia carotovora pv.carotovora Jones, Xanthomonas campestris Pammel pv. phaseoli Smith,Ralstonia solanacearum Smith and a human bacterium Moraxellacatharrhalis.

(D) Screening of Crude Extracts from Agapanthus Africanus for In VitroAntimicrobial and Biostimulatory Activity (1) General

Crude extracts of different plant parts of Agapanthus africanus arescreened in vitro against six plant pathogenic fungi, six plantpathogenic bacteria as well as one human bacterium (see below. The plantparts are dried, ground and extracted with preferably methanol asspecified in more detail in the Examples.

In another approach preparations comprising extracts/dry powders fromAgapanthus africanus in combination with respectively produced extractsfrom wild garlic (Tulbaghia violacea) are tested with respect to theirplant protecting activities.

A standard chemical, Carbendazim/Difenoconazole is used as a positivecontrol. Screening activities are performed using a disk diffusionmethod. To determine the bio-stimulatory activity of crude extracts, twomethods are applied. Firstly, the effect of the extracts on therespiration rate of a monoculture yeast cells is measured using aspecially manufactured respirometer. Secondly, radish seeds are used toascertain the influence of crude extracts on seed germination as well asroot and coleoptile growth in seedlings. In both techniques, ComCat® acommercial biostimulant (EP 1051075) is used as a positive control.

(2) Antimicrobial Properties of A. Africanus Crude Extracts

Crude methanolic extracts of all different plant parts of A. africanussignificantly (P<0.05) inhibit the mycelial growth of all test fungi, invitro, at a concentration of 1 mg/ml (FIG. 1) compared to the standardfungicide, used as a positive control.

The root extract completely inhibits mycelial growth of B. cinerea, S.rolfsii, R. solani and B. dothidea, in vitro, and shows a degree ofcontrol against F. oxysporum (77%) and P. ultimum (64%). A crude leafextract shows a similar inhibitory effect against S. rolfsii, R. solani(FIG. 1) and B. dothidea, slightly lower against B. cinerea (97%) and F.oxysporum (73%) but is equally effective against P. ultimum as was theroot extracts (FIG. 1). Extracts from the stalk also completely inhibitmycelial growth of S. rolfsii and B. dothidea but are slightly lesseffective against B. cinerea (87%) and F. oxysporum (73%) (FIG. 1).

To sum up, a preliminary assessment of the inherent potential of crudeextracts from A. africanus, based on in vitro results, indicates that B.cinerea, S. rolfsii, R. solani and B. dothidea are most sensitive totreatments with extracts from all plant parts. P. ultimum, and to alesser extent F. oxysporum, are more resistant to treatment with thecrude extracts (Table 1).

TABLE 1 In vitro antifungal activity of crude extracts from differentorgans of A. africanus % Mycelial Growth Inhibition for different fungiPlant Plant Botrytis Fusarium Sclerotium Rhizoctonia BotryosphaeriaPythium extract organ cinerea oxysporum rolfsii solani dothidea ultimumPlant X Root 100a ± 0 75c ± 4 100a ± 0 100a ± 0 100a ± 0 63d ± 2 Leaves94ab ± 2 71c ± 2 100a ± 0 94ab ± 3 100a ± 0 64d ± 3 Stalks  90b ± 3 74c± 2 100a ± 0  99a ± 1 100a ± 0 63d ± 2 Flower  98a ± 2 78c ± 5 100a ± 0100a ± 0 100a ± 0 60d ± 4 *Standard 100a ± 0 50e ± 2  51e ± 3  52e ± 2 67d ± 5   7f ± 2

The flower and the aerial part crude extracts also show almost the sameinhibitory effect against all tested fungi, as it is the case for othercrude extracts (FIG. 1). However, aerial part crude extracts are moreeffective in inhibiting the mycelial growth of P. ultimum (79%) thanother plant part extracts when tested separately (FIG. 1). In all casesthe crude extracts out perform the standard fungicide. However, none ofthe extracts exhibit-antibacterial activity against any of the plantpathogenic bacteria tested.

(3) Biostimulator Properties of A. Africanus Crude Extracts

In order to establish whether crude extracts of A. africanus plant partspossess inherent biostimulatory properties, their effect on therespiration rate of a monoculture yeast cells are first determined invitro over a three hour period. Compared to both a water and a positivecontrol (ComCat®), similar respiration rates are observed when extractsof different plant parts are tested separately (FIG. 2). However, theaerial part crude extract increases the respiration rate of yeast cellssignificantly over the first two hours (FIG. 2).

Subsequently, the in vivo effect of crude extracts on the germination ofradish seeds as well as seedling growth is determined. Crude extracts ofthe flowers, flower stalks, leaves and the aerial part crude extractsignificantly (P<0.05) increase seed germination by 21%, 18%, 16% and6%, respectively, compared to the water control (Table 2).

Although most extracts seem to have a stimulatory effect on thegermination of radish seeds in vitro, only the leaf and flower extractsshow a significant stimulatory effect on root growth of the seedlings.Root, flower stalks and the aerial part crude extract, on the otherhand, significantly inhibit root growth compared to the water control(Table 2).

TABLE 2 The effect of crude extracts from different plant parts of A.africanus on the germination of radish seeds as well as seedling growthRoot Coleoptiles Plant extracts Germination (%)† length (mm) length (mm)Roots 62.67 ± 10c 30.62 ± 15.50d 24.01 ± 7.69bc Leaves 71.33 ± 11.5ab43.14 ± 13.61a 24.64 ± 4.12abc Flowers stalks 72.67 ± 5.78a 38.40 ±8.78bc 26.40 ± 4.05ab Flowers 74.78 ± 5.96a 43.09 ± 9.5a 26.71 ± 3.07aAerial part 65.22 ± 4.56bc 35.81 ± 7.29c 23.07 ± 2.37c Comcat 63.11 ±3.45c 40.31 ± 7.96ab 22.12 ± 2.37c Water (distil) 61.67 ± 2.37c 41.06 ±7.46ab 23.10 ± 2.33c †Values designated with different letters, within acolumn, indicate significant differences at the 5% level (P < 0.05)according to Duncan's multiple range procedure.

Both the flower stalk and flower extracts significantly increasecoleoptile growth of radish seedlings in comparison to the watercontrol. The commercial biostimulant, ComCat®, used as a positivecontrol, has no significant effect on either seed germination orseedling growth.

(4) Antifungal Properties of A. Africanus Crude Extracts Combined withExtracts from Tulbaghia Violacea

A crude extract or a dry powder of wild garlic (T. violacea) is preparedanalogously to the methods described here for species of the genusAgapanthus. The extracts or dried powders are mixed in a 1:1 ratio andaquous solutions are applied in different concentrations varying from0.25 mg/ml to 2 mg/ml.

It is interesting to note that a 50:50 mixture of the two extracts,applied at 0.5 mg/ml, shows total control of the six test fungi (Table3), whereas in comparison hitherto, applying separately the two-foldconcentration (1 mg/ml) of the A. africanus preparation or the T.violacea preparation, inhibition of the mycelial growth of the testfungi is not complete. Even a concentration of 0.25 mg/ml of a combinedextract/dry powder preparation (1:1) leads to an over-all inhibition ofthe same fungus system of more than 90%, indicating that significantsynergism is effective in the combination system. The same effect isobservable with other plant-protecting agents.

TABLE 3 In vitro antifungal activity of crude extracts from the aboveground parts of A. africanus (X) and T. violacea (Y) used together in a1:1 ratio and applied at 0.5 mg/ml. Extract Mix % Mycelial GrowthInhibition for different fungi (50:50) Plant Botrytis FusariumSclerotium Rhizoctonia Botryosphaeria Pythium Fungus material cinereaoxysporum rolfsii solani dothidea ultimum Plant X + Above 100a ± 0 100a± 0 100a ± 0 100a ± 0 100a ± 0 100a ± 2 Plant Y ground (50:50) partsStandard 100a ± 0  70c ± 3  68c ± 4  38 ± 2 87bc ± 3  4f ± 1

-   -   Standard broad spectrum fungicide; Carbendazim/difenoconazole        (Eria@)    -   Different letters following values indicate statistical        significant differences.

(5) Summary Results

Results indicate that none of the plant extracts from A. africanus showsany antibacterial activity. However, crude extracts or dry powders ofall different plant parts of A. africanus significantly (P<0.05) inhibitmycelial growth as well as spore germination in all test fungi,indicating a strong antifungal activity of the preparations according tothe invention. Root and flower extracts as well as an extract of theaerial part crude extracts show significantly higher antifungal activitythan extracts from leaves and stalks. Among the tested fungi, Pythiumultimum, and to a lesser extent Fusarium oxysporum, shows a degree oftolerance towards all extracts. This is especially significant in lightof the experience that mycelial growth inhibition by fungicides is moredifficult to accomplish than inhibition of spore germination. Theaverage inhibitory effect of the plant extracts against the test fungiranges between 59 and 100%. Of these the aerial part crude extract(leaves, stalks and flowers combined) is highest (92%), emphasizing thebroad-spectrum fungicidal potential of the extract. Moreover, the potentanti-fungal activity shown by this combined extract indicate asynergistic effect of different active substances and support theassumption of differential accumulation of bioactive compounds indifferent organs of plants. An extract from the aerial part crudeextract of A. africanus as well as a flower extract significantlyenhances the respiration rate of a monoculture yeast cells and all plantpart extracts enhance the germination of radish seeds, thus indicatingthat a bio-stimulatory in vitro activity is effective. The aerial partcrude extract increases the respiration rate of a monoculture yeast cellsubstantially compared to the separate plant part extracts as well as toboth the water control and the positive control, ComCat®. The sameeffect is not observed when the different plant part extracts weretested separately. All the crude extracts of different plant parts of A.africanus as well as the aerial part crude extract increased thegermination percentage of radish seeds indicating a stimulatory effect.It is possible that one or more active substance contained in the crudeextracts could have had a stimulatory effect on one or more of therespiratory enzymes, most probably regulatory enzymes, or even storagematerial mobilization. Combined extracts/dry powders based onpreparations from two or more plants having plant protecting properties,wherein at least one is a species of the genus Agapanthus, show over abroad range antifungal and bio-stimulatory efficacy at least in vitrobased on synergistic effects.

(E) In Vivo Antimicrobial and Bio-Stimulatory Effects of Prepeparationsfrom Agapanthus (1) General

Mycosphaerella pinodes (Berk & Blox.) Vesterger, is a major constraintto field pea (Pisum sativum L.) production and is the most destructiveand widespread disease throughout the field pea growing areas of theworld. All aerial parts of the pea plant are susceptible to infectionwhile growth, yield and seed quality are all adversely affected. Thefungus infects pea seedlings as they emerge causing girdling stemlesions that reduce field pea populations and increase lodging. Later italso causes necrotic lesions on leaflets and stipules and, inexceptional circumstances, abscission of the leaflets. M. pinodes isspread via pycnideospores throughout the season. After germination ofspores, the fungus grows over the plant surface for some distance beforeforming an apersorium and penetrating the cuticle. Symptoms arecharacterized by brown to purplish, coalescing lesions on aerial tissue.Crude extracts of flowers, roots, leaves and the aerial plant parts aresubsequently tested under greenhouse conditions against Mycosphaerellapinodes, the cause of black spot or Ascochyta blight in peas. Fourthinternode leaves are removed from four week old pea plants, placed onmoist filter paper in petri dishes and inoculated with a M. pinodesspore suspension 30 min before and after treatment with the extracts.The control of Ascochyta blight by different concentrations of the crudeextracts from different plant parts of A. africanus is measured in termsof lesion size over a 6 day period at 20° C. in a growth cabinet.

(2) In Vivo Antifungal Activity of Preparations of Agapanthum UnderGlasshouse Conditions

In the in vivo screening antifungal trial, using pea leaves inoculatedwith M. pinodes spores either before or after treatment with the plantextracts, the extract of A. africanus inhibits completely sporegermination of M. pinodes at a concentration near 1 mg/ml, when theextract is applied before spore inoculation. This indicates thatapplication of A. africanus on crops as a preventative measure haspotential in the agricultural industry.

TABLE 4 In vivo antifungal activity of crude extracts from the aboveground parts of A. africanus against Mycosphaerella pinodes on pealeaves Extract Mean lesion Treatment concentration size (mm) %Inhibition Extract sprayed 2 mg ml⁻¹ 0 100% on leaves first and 1 mgml⁻¹ 0 100% spore inoculation 0.5 mg ml⁻¹ 2.37  81% followed 30 min 0.25mg ml⁻¹ 4.07  68% later *Fungicide 0 100% standard 12.8 — Spores onlyLeaves inoculated 2 mg ml⁻¹ 0 100% with spores first 1 mg ml⁻¹ 0.26  98%and extracts 0.5 mg ml⁻¹ 3.8  70% sprayed on leaves 0.25 mg ml⁻¹ 5.29 59% 30 min later *Fungicide 0 100% standard 12.8 — Spores only*Standard broad spectrum fungicide; Carbendazim/difenoconazole(Eria^(©))

Treatment of detached pea leaves with crude extracts of different plantparts of A. africanus, both before and after inoculation with M. pinodesspores, results in significant differences among extracts, extractconcentration and method of inoculation in suppressing lesiondevelopment (Table 4). Among extracts the aerial plant part extract ismost effective in suppressing lesion development, caused by M. pinodeson detached pea leaves, especially when applied before sporeinoculation. The aerial plant part extract is also effective at thelowest concentration (MIC=0.5 mg/ml) compared to other extracts. Whenthis extract is applied after spore inoculation, suppression of lesiondevelopment on pea leaves is also statistically significant compared toother extracts although complete suppression is observed only with thehighest concentration of 2 mg/ml (Table 4)

In comparison the flower extract performs second best in terms of lesiondevelopment suppression both when applied before or after inoculation(MIC between 1 and 2 mg/ml) The root extract completely inhibits lesiondevelopment only at a concentration of 2 mg/ml when applied beforeinoculation. Although complete suppression of lesion development is notobserved with the 2 mg/ml concentration when the root extract is appliedafter spore inoculation, the degree of suppression is statisticallysignificant compared to the untreated control, except at 0.25 mg/ml(Table 4). The leaf extract fails to suppress lesion developmentcompletely both when applied before and after spore inoculation but, inboth cases, the degree of suppression obtained is significant comparedto the untreated control, except at 0.25 mg/mg (Table 4).

Crude extracts of different plant parts of A. africanus suppress in vivolesion development on detached pea leaves to variable degrees dependingon the concentration applied as well as the time of inoculation. Ofthese the aerial part crude extract is most effective at allconcentration levels tested, compared to the other plant part extracts,both when applied before and after inoculation of detached pea leaveswith M. pinodes spores. The flower extract also shows significantsuppression of lesion development at a relative low concentration. Asthe aerial plant part extract contains compounds from flowers, flowerstalks and leaves, the possibility of different active substancescontained in the different parts showing a synergistic effect in eitherinhibiting spore germination or mycelial infection or both is notexcluded.

The ability of the aerial plant extract as well as the flower extract tocompletely suppress lesion development even when applied afterinoculation of detached pea leaves, is especially significantconsidering that the standard fungicide failed to do so.

Treatment of detached pea leaves with root and leaf extracts is lesseffective in preventing M. pinodes infection at lower concentrationswhen applied both before and after spore is inoculation compared to theaerial plant part and flower extracts. The necrotic lesions measured onpea leaves treated with root and leaf extracts at concentrations lowerthan 1.0 mg/ml are similar to that measured on control leaves inoculatedwith spores only. However, when applied to detached pea leaves beforespore inoculation, both extracts still show significant suppression oflesion development. Interestingly, the aerial plant part extract, ofwhich leaves formed the largest portion, performs best overall. Apossible synergistic effect between compounds contained in flowers,flower stalks and leaves in enhancing the fungicidal properties of A.africanus again is assumed. In terms of the potential to develop anatural product from A. africanus, the fact that the root extract isless effective than the aerial plant part extract underlines itsexclusion and implies non-destructive collection.

The present study confirms that, especially a combined crude extract ofaerial plant parts of A. africanus at a concentration of 0.5 mg/ml andlower, has the potential to be applied as both a preventative orcorrective measure against infection of pea plants by M. pinodes spores.There are strong indications that the extract possesses significantpotential as a corrective broad spectrum antifungal agent. Inconclusion, the efficacy of different plant part extracts of A.africanus varies in suppressing lesion development on detached pealeaves caused by M. pinodes in vivo. The aerial plant part extract ismost effective, especially when applied before spore inoculation and ata relatively low concentration of 0.5 mg/ml. However, application athigher concentrations after inoculation with M. pinodes spores showscomplete inhibition of spore germination or infection or both.Importantly, none of the extracts causes phytotoxic yellowing ornecrosis on detached pea leaves even at the highest concentrationsapplied.

(3) Phytotoxic Effects of Preparations from Agapanthus on Pea LeavesUnder Glasshouse Conditions

The in vivo phytotoxicity rating of the aerial plant parts, flower, rootand leaf crude extracts of A. africanus, in terms of its interactionwith and potential to induce necrosis in pea leaves, reveals that thecrude extract is not phytotoxic even at the highest concentration tested(Table 5a, b) and the symptomless effect of the extract is similar tothat of the water and standard fungicide controls. All plant partextracts of A. africanus as well as the standard fungicide controldiffers significantly from the leaf necrosis induced by the M. pinodesspore suspension.

TABLE 5a Mean foliar phytotoxicity symptom rating on a six-categoryscale following direct inoculation of fourth node pea leaflets with thehighest concentration of crude aerial plant part, flower roots and leafof A. africanus. Mean foliar phytotoxicity symptom Plant extractsapplied as foliar treatments Concentration at 2 mg/ml Aerial plant parts0.0b Flowers 0.0b Roots 0.0b Leaves 0.0b Standard fungicide 0.0b Sporesuspension 4.2 ± 0.8a

TABLE 5b Phytotoxic effect of crude extracts from A. africanus plants onpea leaves. Mean Lesion size Extract (mm) indicating TreatmentConcentration phytotoxicity Crude extract only 2 mg ml⁻¹ 0 1 mg ml⁻¹ 00.5 mg ml⁻¹ 0 0.25 mg ml⁻¹ 0

None of the different plant part extracts of A. africanus show anyphytotoxic effect on detached pea leaves even at the highestconcentration applied.

(4) Control of Sorghum Covered and Loose Smuts by an Aerial Part CrudeExtract of Agapanthus Africanus Under Field Conditions

Sorghum (Sorghum bicolor L. Moench) is an important source of food inmany non-developed countries and serves as staple food for the majorityof people. It is predominantly grown in small-scale production systemsunder a wide range of environmental conditions. However, production ofsorghum is less than 1.0 ton/ha due to various reasons. Sorghum coveredkernel (fSporisorium sorghi Link, G. P. Clinton) and loose kernel smuts(Sporisorium cruenta Kuhn, A. A. Potter) are major factors that accountfor low yields. Both diseases occur frequently where sorghum is grownwithout treating seeds against these two pathogens.

Treatment of sorghum seeds with an aerial part crude extract of A.africanus before planting completely (100%) (P<0.05) reduce theincidence of both covered smut (Table 6a) and loose smut (Table 6b)compared to the corresponding untreated controls, and in both casescompared favourably with the synthetic fungicide Thiram.

TABLE 6a Effect of an aerial part crude extract of A. africanus on thepercentage covered kernel smut disease incidence under field conditions.Mean plant % mean smut Yield Treatments population incidence (ton ha⁻¹)Aerial plant extract 171 ± ?a 0b 3.0a Thiram 175a 0b 2.6ab Control 173a5a 1.6b

TABLE 6b Effect of an aerial part crude extract of A. africanus on thepercentage loose kernel smut disease incidence under field conditions.Mean plant % mean smut Yield Treatments population incidence (ton ha⁻¹)Aerial plant extract 175 ± ?a 0b 2.9a Thiram 175a 0b 2.1ab Control 175a18a  1.3b

Values designated with different letters differed significantly (P<0.05)according to Duncan's Least Significant Difference (LSD) statisticalprocedure.

Inoculation of pre-planted sorghum seed with covered or loose smutsspores, without also treating the seeds with either Thiram or the crudeA. africanus extract (untreated controls), significantly decreases thefinal yields (Tables 6a, b). In the case of covered smut the yield lossis 46.7% and, in the case of loose smut, 55.2%. However, in both cases,there is no significant difference in yield between plots treated witheither Thiram or the A. africanus crude extract. Probably due to thehigh standard deviation, no significant difference in yield between theThiram treated and untreated controls can be observed in both cases.

The percent covered and loose smuts incidences are negatively correlated(R²=−0.92 and −75 respectively) with sorghum grain yield indicating thenegative impact both smut diseases had on the yield.

(5) The Effect of A. Africanus Extracts on the Defense Mechanism ofPlants (SAR)

Plants (e.g. wheat and sunflower) elicit, when treated with an extractof A. africanus and another reference plant (Tulbaghia violacea)according to the invention, a significant activation of PR-proteins suchas NADPH oxidase, peroxidase and β-1,3-glucanse. Wheat plants treatedwith the A. africanus extract show strong induction in NADPH oxidaseactivity after 6 h reaching the highest activity at 9 h (112%) over theprevious sampling time. Activity remained high for up to 48 h (FIG. 4).Sunflower reacts to treatment with the A. africanus extract in the sensethat two peaks in NADPH oxidase activity can be observed. The first peakis reached 6 h after treatment with an increase in activity of 61% overthe previous sampling time while the second peak in activity is reached48 h after treatment with an increase in activity of 333% over theprevious sampling time (FIG. 5). From these results it seems that in theC4 plant, wheat, activity induction by treatment with the A. africanusextract is more pronounced than in sunflower, a C3 plant. Wheat treatedwith the A. africanus extract shows a significant induction (100%) inperoxidase activity 24 h after treatment and this activity is maintainedover the test period (FIG. 6). In the case of sunflower the A. africanusextract induces peroxidase activity significantly especially after 48 hand 96 h (FIG. 7). For A. africanus the induction is 212% after 48 h and230% after 96 h. The sunflower control, however, shows a slight increasein peroxidase activity over the 96 h period indicating some naturalresistance. Agapanthus extracts induce defense mechanisms in wheat andsunflower plants. These extracts induce localized acquired resistance,the accumulation of PR-proteins by gene activation and ultimatelysystematic acquired resistance. The fact that the extracts induce adefense response in both the wheat and sun flower samples indicate thatthe extracts are responsible for the induction of a generalbroad-spectrum defense response. The extract-induced increase in defenserelated enzyme activities was lower, but comparable to the increaseobtained during infection with resistant cultivars

(F) Isolation, Purification and Identification of Antifungal Compoundsfrom Root and Aerial Plant Part Extracts of Agapanthus Africanus

(1) General

Although information about the chemical analysis of different plantparts of A. africanus is scanty, initial attempts was made by Takeda etal. (1955, Chemistry Abstract 50) who isolated and identified thecompound yuccagenin from the roots. Others (Stephen, 1956 JournalChemistry Society 1167.; Mathew et al., 1957 Journal Chemistry Society262), working with several unspecified species of Agapanthus, reportedthe new spirostan sapogenin, agapanthagenin. Subsequently, in additionto the previously reported compounds, Gonzalez et al. (1973Phytochemistry 13:627-631) isolated and identified two new spirostansapogenins from the root system of A. africanus. Most previous studiesconcentrated on the isolation and identification of natural compoundsfrom the root parts of A. africanus, but the relationship between thesecompounds and antimicrobial activity has not been established. Moreover,virtually nothing is known about the chemical composition of the aerialplant parts as well as their fungicidal properties.

(2) Antimicrobial Activity of Liquid-Solid Extractions of the Roots andAerial Plant Parts

The semi-purified fractions of different plant parts of A. africanus,obtained by means of liquid-solid extraction, differ significantly ininhibiting the mycelial growth of F. oxysporum (Table 8). Thesemi-purified extract of the roots, contained in diethyl ether, and boththe ethyl acetate and dichloromethane extract of the aerial plant parts,significantly (P<0.05) inhibit mycelial growth of F. oxysporum comparedto the hexane extract (Table 8). The diethyl ether root fraction showedthe highest inhibition (62%) compared to the ethyl acetate anddichloromethane fractions that showed similar inhibition effects (51%).

In case of the combined aerial plant part extract, both the ethylacetate and dichloromethane semi-purified liquid-solid extracts are mostactive and completely inhibit the mycelial growth of F. oxysporum (Table7). This is statistically significant compared to the antifungalactivity of both the hexane and diethyl ether fractions and comparedfavorably with the standard fungicide, Carbendazim/difenoconazole.Mycelial growth inhibition of F. oxysporum by semi-purified fractions ofthe aerial plant parts is also significantly (P<0.05) higher than thatof the roots (Table 7).

TABLE 7 Antifungal activity of semi-purified liquid-solid extractions ofthe roots and aerial parts of A. africanus against Fusarium oxysporiumMycelial growth inhibition (%)† Plant part Solvent Roots Aerial partsHexane 24.3d  5.4c Diethyl ether 62.3b 13.7b  Ethyl acetate 51.1c 100aDichloromethane 51.3c 100a Fungicide 100a   100a †Values designated withdifferent letters within a column indicate a statistically significantdifference at the 5% level (P < 0.05) according to Duncan's multiplerange procedure.

The recovered yields of the root and aerial plant part liquid-solidextractions are presented in Table 8. Despite its low activity, thehexane solvent system provides high amounts of semi-purified residue ofthe roots and aerial plant parts ranging between ca. 4.3 to 5.4% whilethe diethyl ether solvent system provides ca. 3% and 2% from the rootsand aerial plant parts, respectively. The ethyl acetate solvent systemyields approximately ca. 1% residues in both the root and aerial partextracts while the recovered yield from the dichloromethane solventsystem is less than ca. 1% in both cases.

TABLE 8 Residual yield recovered from A. africanus root and aerial plantpart extracts obtained by means of liquid-solid extraction with a seriesof solvents, after drying at 35° C. Crude root extract Crude aerial partextract Solvents (268.5 g) (368.83 g) Hexane 14.5 g 15.89 g  Diethylether  7.6 g 5.96 g Ethyl acetate 3.09 g 4.33 g Dichloromethane 0.36 g0.45 g The original dry mass of crude extracts is indicated in brackets.

The diethyl ether extract of the root and the ethyl acetate as well asthe dichloromethane fractions of the aerial parts show the highest(>50%) antifungal activity against F. oxysporum. However, as therecovery of compounds in the dicholoromethane fraction is extremely low(Table 8), only the ethyl acetate fraction of the aerial parts, togetherwith the diethyl ether fraction of the roots, are chosen for furtheractivity directed column chromatography fractionation.

(3) Activity Directed Column Chromatography Fractionation of the MostActive Liquid-Solid Extracts

After collecting 300 column chromatography fractions of the diethylether root extract, every third fraction is spotted on a Q-TLC plate anddeveloped with butanol:acetone:methanol (7:2:1; v/v) in order to obtainTLC profiles used as an indicator for combining fractions with similarprofiles. In this way 17 combined column fractions can be obtained fromthe root extract of which nine showed high mycelial growth inhibition(65-97%) against F. oxysporium (Table 9). After treating the ethylacetate aerial part fraction in the same way, 20 combined columnchromatography fractions are obtained of which six were active ininhibiting the mycelial growth of F. oxysporium by more than 50% (Table9).

Although nine of the combined column chromatography root fractions showhigh mycelial growth inhibition against F. oxysporum at a relatively lowconcentration of 625 μg/ml (w/v), only fraction 13 is used for furtherpurification by means of preparative thin layer chromatography (PTLC)due to the extremely low recovery of the other fractions. In the case ofthe aerial plant parts, only column fraction 14, that showed completemycelial growth inhibition against the test organism at the lowconcentration of 125 μg/ml (w/v), was further purified (Table 9).

TABLE 9 Antifungal activity of combined fractions obtained from the mostactive root and aerial plant part liquid-solid extracts following columnchromatography against F. oxysporum. Combined column % mycelial growthinhibition of Plant part fraction number F. oxysporum Roots 8 76 9 67 1093 11 97 12 83 13 83 14 74 16 65 17 71 Aerial plant parts 7 58 8 60 14100 17 57 18 55 19 53 Only the most active combined columnchromatography fractions are shown.(4) Preparative Thin Layer Chromatographic (PTLC) Purification of ActiveCompounds from Column Chromatography Fractions

Following preparative thin layer chromatography purification of activecolumn fraction number 13 obtained from the root, 12 purified P-TLCfractions can be recovered, of which fraction 9 is most active (95%)against F. oxysporum (Table 10). In the case of active column fractionnumber 14 obtained from the aerial parts, three purified P-TLC fractionsare recovered following washing with acetone, of which fraction number 1shows complete mycelial growth inhibition against F. oxysporum (Table10).

TABLE 10 Antifungal activity of P-TLC fractions obtained from root andaerial plant parts against F. oxysporum. Mycelial growth inhibition (%)P-TLC fractions against F. oxysporum Fraction 9 of the root 95 Fraction1 of the aerial plant parts 100

After controlling the most active P-TLC fractions of both the root andaerial parts for purity, by obtaining Q-TLC profiles after acetylatingthe molecules and acidifying the mobile phase with 1N HCl, the rootfraction consists of four compounds (Table 11). By means of acidifiedP-TLC separation, these four compounds are purified and tested forantifungal activity. All four compounds are highly active. The activeP-TLC fraction of the aerial parts proves to contain only one purecompound that is active (Table 11). All five of these pure compounds aresubsequently subjected to nuclear magnetic resonance (NMR) spectroscopyin order to elucidate their molecular structures.

TABLE 11 Antifungal activity of pure compounds obtained from the mostactive P-TLC root and aerial plant part fractions, followingacidification, against F. oxysporum. % mycelial growth inhibition ofPlant part Compound number F. oxysporum Roots 1 100 7 87 8 93 9 97Aerial plant parts 1 100(5) Identification of Active Compounds Purified from Roots and AerialParts of A. Africanus by Means of Nuclear Magnetic Resonance (NMR)Spectroscopy

Based on the ¹H NMR spectra the single antifungal substance derived fromthe combined aerial plant parts of A. africanus provides a novelcompound, saponin (1). Exactly the same saponin (1) can be identified asone of the four active substances derived from the roots of A. africanustogether with three known flavonoids, 5,7,4′-trihydroxyflavanone (7),5,7,3′4′-tetra-O-acetylflavanone (8) andtrans-4,2′,4′-Tri-O-acetylchalcone (9). Structural elucidation can beachieved via spectroscopic methods (1D NMR and 2D NMR) FAB and EI-MSspectrometry, and chemical methods such as hydrolysis.

(6) Saponin (1) Isolated from Both the Roots and Aerial Parts of A.Africanus

The methanol extract from the roots and aerial plant parts of A.africanus yields compound (I) as a light brown precipitate in relativelylarge amounts. To obtain an acceptable level of purity, the fractionsare washed repeatedly with acetone. This, and the highly insolublenature of compound (1) (FIG. 3A), invariably lead to substantial losses,prohibiting reliable quantification. Due to the complexity of the ¹H NMRspectrum of the non-derivatised saponin, the peracetate derivative (2)(FIG. 3B) is used in the structural elucidation. FAB-MS shows the [M+H]⁺ion at m/z 448, consistent with the molecular formula C₂₇H₄₄O₅ of theaglycone with the molecular mass of 448.

(7) Isolation and Identification of Flavones

In addition, flavanones (compound 7) and (compound 8) can be isolatedfrom the roots after acetylation of fractions nine of the diethyl etherextract by means of P-TLC chromatography. Characteristic of thesecompounds is the presence of the 3-CH₂ [(two doublets of doublets, δ(3.00-3.15) and (2.70-2.85)] and the 2-H [(doublets of doublets, δ(5.00-6.00)] in their ¹H NMR spectra.

Besides the sapogenin compound isolated from the root parts of A.africanus, by far the most frequently encountered flavanone, naringenin(5,7,4′-trihydroxyflavanone), was identified

It is common as a free phenol, occurs with a wide variety ofglycosylation patterns, has been isolated in all of its possibleO-methylated forms and is susceptible to various C-alkylation processes(Batterham et al., 1964). Naringenin can be isolated after acetylationand P-TLC separation as the 5,7,4′-tri-O-acetyl derivative (7).

Additionally, 5,7,3′4′-tetra-O-acetylflavanone (8) can be isolated afteracetylation and PLC separation from fraction number nine of the rootpart.

Moreover, an additional compound, trans-4,2′,4′-Tri-O-acetylchalcone,Isoliqiuritigenin can be isolated as a peracetate derivative (9) afteracetylation and PLC separation from fraction-number nine of the roots.This compound is found in many leguminous plants (Roux et al., 1962,Biochemical Journal, 82:324).

5

(8) Discussion

Subsequent activity directed purification of only the most activefractions, using column and preparative thin layer chromatographyfollowed by nuclear magnetic resonance (NMR) spectroscopy, and fast atombombardment (FAB-MS), reveals the universal presence of a novel saponin(1) with strong antifungal activity at a concentration of approximately125 μg/ml (100-150 μg/ml). Additionally, three flavonoids5,7,4′-tri-O-flavanone (7), 5,7,3′4′-tetra-O-acetylflavanone (8) andtrans-4,2′,4′-Tri-O-acetylchalcone (9), showing strong antifungalactivity at a concentration of about 625 μg/ml (570-650 μg/ml), can bepurified from A. africanus roots.

Semi-purified extracts of the roots and aerial parts of A. africanusobtained by means of solid-liquid extraction with hexane, diethyl ether,ethyl acetate and dichloromethane solvent systems in this order ofincreasing polarity, variety in antifungal activity. The diethyl etherextraction of the roots is most active in inhibiting the mycelial growthof F. oxysporum, used as test organism in the activity directedpurification protocol, while the ethyl acetate extraction of the aerialparts is most active. Although the hexane extraction removes mostcompounds from both the roots and aerial parts, it is comparably activein both cases.

Following column fractionation of the active liquid-solid extractions,the Q-TLC profiles show diverse chemical constituents in the roots andthe aerial plant parts of A. africanus while the latter extract containscomparatively more compounds. However, four active compounds can beisolated from the diethyl ether root extract while only one activecompound can be detected in the ethyl acetate aerial part extract.Despite the lower number of active substances in the aerial parts,following different extraction procedures, this fraction is more activein inhibiting the mycelial growth of the the test fungus, F. oxysporum.

Compounds purified from the roots and aerial plant parts of A. africanuscan be identified by means of ¹H-NMR and ¹³C-NMR spectroscopy. The majorcompound predominantly isolated from both the roots and aerial plantparts is a novel steroidal saponin with a three sugar chain attached atthe C3 position of ring A in the aglycone moiety. The compound can beidentified as3-[O—β-D-glucopyranosyl-(1″-3′)-α-L-rhamnosyl-(1″-2′)-β-D-glucopyranosyloxy]agapanthegenin. Previously, a C-3 monoglycosylated saponin, with thesame aglycone, was isolated from the root system of A. africanus by bothStephen (1956) and Gonzalez et al. (1974). However, the saponinidentified in this study is different with respect to the additionallyattached three sugar chain. Sapogenins with a three sugar chain attachedare of sporadic occurrence. Additionally, three flavonoids with notablefungicidal activity, 5,7,4′-tri-O-flavanone,5,7,3′4′-tetra-O-acetylflavanone and trans-4,2′,4′-tri-O-acetylchalcone,can be isolated from the roots of A. africanus. Each individual purifiedflavonoid showed significant in vitro mycelial growth inhibition againstF. oxysporum.

The in vitro and in vivo antifungal activity observed with crude andsemi-purified extracts of different plant parts of A. africanus seems tobe related to the presence of the major compound, a steroidal saponin inall plant parts, as well as the presence of the three flavonoids5,7,4′-tri-O-flavanone, 5,7,3′4′-tetra-O-acetylflavanone andtrans-4,2′,4′-Tri-O-acetylchalcone in the root system.

In summary, all four compounds in their pure forms provide an acceptablelevel of efficacy in inhibiting the mycelial growth of the test fungusin vitro and hold great promise to be applied as one or more naturalproducts in integrated disease management systems in vivo. However, dueto a possible synergistic effect of the combined compounds, theapplication of either a crude or a semi-purified extract might beconsidered. Importantly, in light of the fact that A. africanus is aperennial, an extract of the combined aerial parts might possess themost potential to be developed into a natural product as harvesting ofthe above soil parts is non-destructive. This implies that the potentialexists for A. africanus to be cultivated as a new crop and to serve as asource for a natural fungicide with broad spectrum control ofeconomically important plant pathogens, especially to small-scalefarmers who have no access to synthetic chemicals.

FIGURE LEGENDS

FIG. 1: In vitro inhibitory effect of crude extracts from differentplant parts of A. africanus on the mycelial growth of various fungi.Vertical bars indicate standard deviations. Bars designated withdifferent letters indicate significant (p<0.05) differences betweenmeans according to Duncan's multiple range procedure. Y-axis: mycelialgrowth inhibition (%). (1)=root; (2)=leaves, (3)=Flower stalks;(4)=Flowers; (5)=above ground plant parts; (6)=reference fungicide

FIG. 2: The effect of crude extracts from different plant parts of A.africanus on the respiration rate of a monoculture yeast cells. Verticalbars indicate standard deviations. X-axis: time (min); Y-axis:respiration rate (cm³ CO₂ release).

1=roots, 2=flower stalks, 3=above ground plant parts, 4=water, 5=leaves,6=flowers, 7=ComCat®

FIG. 3A: Sructure of novel saponin (1):

3-[{O—O-D-glucopyranosyl-(1″-3′)-α-L-rhamnosyl-(1″-2′)}-β-D-glucopyranosyloxy]agapanthegenin.

1: R═H; 2:R=Ac

FIG. 3B: Structure of the aglycone (3; agapanthegenin), the glucosylatedsapogenin (5) and their respective O-acetyl derivatives (4 and 6).

3: R═H; 4: R=Ac; 5: R=glucose; 6: R=acetyl glucose

FIG. 4: NADPH oxidase activity pattern in wheat treated with anAgapanthus extract and a Tulbaghia extract (as reference) according tothe invention in dependency of the time after treatment.

FIG. 5: NADPH oxidase activity pattern in sunflower treated with anAgapanthus extract and a Tulbaghia extract (as reference) according tothe invention in dependency of the time after treatment.

FIG. 6: peroxidase activity pattern in wheat treated with an Agapanthusextract and a Tulbaghia extract (as reference) according to theinvention in dependency of the time after treatment.

FIG. 7: peroxidase activity pattern in sunflower treated with anAgapanthus extract and a Tulbaghia extract (as reference) according tothe invention in dependency of the time after treatment.

EXAMPLES Example 1 Preparation of Crude Extracts

Dried plant material was powdered, using a Retsch SM2000 cutting milland soaked in 100% methanol (v/g) at a ratio of 2 ml g⁻¹ dry weight on aroller mill overnight and the supernatant subsequently decanted. Thiswas repeated five times. The combined suspensions were filtered twice,first under vacuum through a double layer of Whatman filter paper (No. 3and No. 1) and then by gravity through a single sheet of Whatman No. 1filter paper. The methanol was removed from the clear supernatant bymeans of vacuum distillation at 30-35° C. using a Büichi RotaryEvaporator. The remaining aqueous solution was referred to as the crudeextract.

Example 2 Preparation of a Dry Powders

Instead of the preparation of a crude extract according to Example 1,the preparation of a dry powder is also applicable. The implication forit is that a considerable reduction in production costs might beachieved and that more hectares of cultivated land can be treated withthe product in this form. The preparations of Example 1 and Example 2show almost identical qualitative and quantitative results with respectto their plant protecting activity/efficacy. Plant material is dried at35° C., preferably in a drying oven. Dried plant material is firstground to a course powder, using a Retsch SM2000 cutting mill, andsubsequently to a fine powder using a special mill than can grind toparticles smaller than 100 micron to prevent clogging in a nozzle spraysystem. The powder is soaked in 100% methanol or ethanol (v/g) at aratio of preferably 2 ml/g dry weight on a roller mill for 48 h and thebulk of the methanol decanted before the remaining methanol is allowedto evaporate on a large surface. Subsequently, the powder is treatedwith 100% Ethanol for 24 h in exactly the same way as with methanol. Thefinal product is in the form of a wettable powder that is applied at arate of preferably 1 g/l and at approximately 300-600 liters perhectare.

Example 3 Screening for Antifungal Properties

A modified agar dilution method (Rios et al. 1988, Journal ofEthnopharmacology 23:127-149) was used for determining the inhibition ofmycelial radial growth of the test organisms by the plant extracts. Allplant pathogenic test fungi were cultured on 2% (rn/v) malt agar,prepared according to the specifications of the manufacturers, andautoclaved for 20 min at 121° C. On cooling to 45° C. in a waterbath,300 μl of a 33% (m/v) Streptomycin solution was added to the basalmedium for controlling bacterial growth. Dried material of each plantextract was dissolved in 100 ml sterile distilled water and amended inthe agar to yield a final concentration of 1 mg/ml. Working in a laminarflow cabinet, the medium was poured into 90 mm sterile plastic Petridishes, to a thickness of 2-3 mm, and allowed to set The center of eachtest plate was subsequently inoculated with a 5 mm size plug of 7-10 dayold cultures, for each of the pathogens separately. A plate containingonly the basal medium served as control. Additionally, a platecontaining a standard fungicide, carbendazim/difenococnazole(Eria®-187.5 g/1 EC), at 1 μg/ml was used as a positive control againsteach test organism separately to determine the effectiveness of theextracts by comparison. Plates were incubated for four days at 25±2° C.in a growth cabinet. Each assay was performed in triplicate. Radialmycelial growth was determined after four days by calculating the meanof two perpendicular colony diameters for each replicate. Themeasurement included the assay wells (March et al., 1991,Zentralbladtfur Mikrobiologie 146:291-295; Pfaller et al., 1992,Antimicrobial Agents and Chemotherapy 36:1805-1809) and was expressed aspercentage mycelial growth inhibition by calculating according to theformula of Pandey et al (1982, Zeitschrift Pflanzenkrankheit undPflanzenschutz 89:344-349): (dc−dt)/dc×100, where dc=average diameter ofthe fungal colony of the negative control and dt=average diameter of thefungal colony treated with the extracts. The data reported were pooledfrom the two experiments.

Example 4 Screening for Antibacterial Properties

A modified agar diffusion method (Caceres et al., 1993, Journal ofEthnopharmacology 38:31-38) was used. Plate count agar (PCA, Biolab) wasprepared in the same way as malt agar used in the antifungal screeningtests except that the plant extracts were not suspended in the agar.Mother cultures of all plant pathogenic bacteria were sustained onnutritional agar (Caceres et al., 1991, Journal of Ethnopharmacology31:193-208, Rasoanaivo et al., 1993). Overnight soup cultures of thetest bacteria were initially prepared separately in sterile 1% (w/v)nutrient broth (Biolab) solutions at 30° C. (Meyer et al, 1995). Onehundred μl of each of these bacterial suspensions were subsequentlyseparately transferred to 90 mm Petri dishes containing the sterile PCAagar and evenly streaked on the surface using sterile swabs. Petridishes were divided into four quarters and a hole, 6 mm in diameter,plunged into the agar of each quarter by means of a sterile cork borer.50 μl of the 1 mg/ml crude stock solution extracts were transferred intothe holes in the agar. The plates were equilibrated at 4° C. for 1 h toallow the extracts to diffuse into the agar before incubation commenced.In this way the development of clear inhibition zones was optimized.Plates were incubated for three days at 25° C. for all plant pathogenicbacteria, but at 35° C. for M. catharrhalis. Each assay was performed induplicate. Inhibition zones were measured using a digital caliper.

Example 5 In Vitro Screening for Bio-Stimulatory Properties of CrudeExtracts

Two methods were applied to determine the biostimulatory potential ofthe organ crude extracts of A. africanus.

Method 1: Manometric Method for Determining the Effect of Crude Extractson the Respiration Rate of a Monoculture Yeast Cells

A specially constructed glass respirometer with a short bulged section(reservoir) to contain the yeast cells and a long calibrated tube,closed at the top end to collect CO₂ gas, was used in determining theeffect of the A. africanus crude extracts on the respiration rate of amonoculture yeast cells. Dry baker's yeast (0.8 g) was placed in thereservoir of the respirometer. Subsequently, 70 ml of each of the plantextracts, previously prepared at a concentration of 0.5 mg/ml andcontaining 5 mg/ml glucose to serve as respiratory substrate for theyeast cells, was added to the respirometer. The apparatus was tiltedsideways to release air bubbles trapped in the dry baker's yeast andplaced in a water bath pre-heated to 29° C. ComCat®, a commercialbiostimulant, was used as a positive control at 0.5 mg/ml (optimumconcentration according to the manufacturers; Agraforum, Germany, 2002)and distilled water as a second control. CO₂ release by the yeast cellswas measured in cm³ at 30 minute intervals over a three hour incubationperiod by reading the released gas volume from the calibrated tube.Tests were performed in triplicate.

Method 2: the Effect of Different Organ Crude Extracts of A. Africanuson the Percentage Germination of Radish Seeds and Subsequent SeedlingGrowth

Two sheets of special germination paper (30×30 cm) were used to test theeffect of each plant crude extracts of A. africanus on the germinationof radish seeds as well as the subsequent seedling growth. A line, 10 cmfrom the top, was drawn on the one sheet and 20 radish seeds spacedevenly on the line. A second sheet of germination paper was placed ontop of the first and moistened with either 0.5 mg/ml solutions of thecrude extracts, distilled water (negative control) or 0.5 mg/ml solutionof ComCat® (positive control). Both sheets of paper were rolled uplongitudinally and placed upright in Erlenmeyr flasks containing eithercrude extract, distilled water or the ComCat® solution and kept at 25°C. in a growing chamber in the dark. Seed germination as well ascoleoptile and root lengths were determined at 24 h intervals over a 96h incubation period. Tests were performed in triplicate.

Example 6 Statistical Analysis of Data

Analysis of variance (ANOVA) was performed on the data, using the SAS(1999; SAS/IML software; Version 6; SAS Institute) program, to identifydifferences between treatments. Duncan's multiple range (DMR) procedurefor comparison of means (Steele & Torrie, 1980, Principles andprocedures of statistics, 2^(nd) Edition. New York: McGraw Hill.) wasapplied to separate means (P<0.05).

Example 7 Isolation of Mycosphaerella Pinodes

M. pinodes was isolated from diseased leaves and stems of various wintercultivars of field pea at the time of senescence. Collections of theinfected plant material were made from the central and south easternpea-growing areas of Ethiopia. Pieces of the diseased tissues weresurface sterilized for 1 minute in 96% (v/v) ethanol, 3 minutes in a3.5% (v/v) NaCl solution (Moussart et al., 1998, European Journal ofplant Pathology 104:93-102) and 30 seconds in 96% (v/v) ethanol. Thetissues were subsequently aseptically transferred to corn meal agaramended with streptomycin (0.3 ml/l) in 9 cm Petri dishes and incubatedat 20±1° C. in a growth chamber. Isolates initially obtained from theplant material were then grown on Coon's medium (Ali et al., 1978,Australian Journal of Agricultural Research 29:841-849) consisting of 4g maltose, 2 g KNO₃, 1.2 g MgSO₄, 2.7 g KH₂PO₄ and 20 g agar. Cultureswere incubated for 14 days to obtain pycnidiospores. To obtain anisolate derived from a single uninucleate cell, a suspension ofpycnidiospores was streaked on 15% water agar, incubated overnight at20±1° C. and examined under a dissecting microscope (80× magnification).A germ tube arising from one cell of a pycnidiospore was severed andtransferred to Coon's agar (Clulow & Lewis, 1992, Plant Pathology41:362-369). Six isolates of M. pinodes were obtained. All isolates froma single-spore and cultures were maintained on Coon's agar slants andstored in the dark at 5° C.

Example 8 Preparation of a M. Pinodes Spore Suspension

Oat meal agar was prepared by gently heating 30 g of oats in 1 litredistilled water for 1 h, stirring frequently, and subsequently filteringthrough a fine sieve upon which the volume was readjusted to 1 litre.Twenty g of technical agar and 0.1 g Keltane AP was added to thefiltrate to yield a 2% (m/v) agar concentration. The agar was autoclavedfor 15 min, poured into Petri dishes and allowed to cool off beforeinoculation of three oatmeal plates with M. pinodes mycelia. Plates wereincubated in a 12 h photoperiod incubator at 20° C. for 14 days, toensure the production of pycnidiospores. To prepare the inoculum (sporesuspension), sterile distilled water was added to the 14-day-oldcultures dislodging spores gently with a sterile glass rod. Thesuspension was subsequently filtered through four layers of cheese clothin order to remove the mycelia and the concentration of pycnidiosporeswas determined by means of a haemocytometer. The pycnidiosporeconcentration was adjusted to 1×10⁵ spores per ml (Nasir & Hoppe, 1997,Annals of Applied Biology 18:32-33) with sterile distilled water priorto the inoculation of pea leaves.

Example 9 In Vivo Assessment of Crude Extract Phytotoxicity

Pea seeds were planted in plastic pots in Bainsvlei soil and grown in aglasshouse (minimum temperature 18° C.). Four weeks after planting, whenthe leaflets on the third and fourth nodes were fully expanded, threefourth node leaflets per replicate were removed from the plants, placedon Schleicher and Schull No. 595 filter paper and moistened with 4 ml ofsterile distilled water in 9 cm Petri dishes. 30 μl of each of a 0.25,0.5, 1.0 and 2.0 mg/ml solution of the crude extract were placedseparately on each of the three leaves per Petri dish and replicatedthree times. Treatment of the leaves with water and a standard fungicide(Carbendazim/difenoconazole) served as controls. Petri dishes containingthe treated leaflets were incubated at 20° C. in a day/night incubatorprogrammed for a 16 h day cycle while 2 ml sterile distilled water wasadded daily to keep the filter paper moistened. Six days aftertreatment, phytotoxicity symptoms were assessed on leaves using asix-category scale [0=symptomless; 1=<5% necrotic flecks; 2=>5% necroticflecks; 3=<50% of inoculated area necrotic; 4=50-100% of inoculated areanecrotic; 5=necrosis spreading beyond inoculated areas] based on stereomicroscopic observations (Clulow et al., 1991, Mycological Research 95:817-820).

Example 10 In Vivo Assessment of Crude Extract Antifungal PropertiesUnder Glasshouse Conditions

Fourth node pea leaflets were obtained and sustained on moist filterpaper in Petri dishes as described for the phytotoxicity assessmenttest. In vivo control of M. pinodes spore infection of the leaves bydifferent concentrations (0.25, 0.5, 1.0 and 2.0 mg/ml) of the aerialplant parts, roots, leaves and flowers of A. africanus was followed intwo ways namely, by inoculating the leaves with 15 μl of a sporesuspension (1×10⁵ spores/ml; Nasir & Hoppe, 1997, Annals of AppliedBiology 18:32-33) 30 min before applying the different concentrations ofthe crude extract separately, and the other way around. A standardfungicide, carbendazim/difenoconazole, currently used against Ascochytablight in peas, as well as leaves inoculated only with the sporesuspension, served as controls. Three leaves per Petri dish representeda replicate and the experiment was performed in triplicate. Petri dishescontaining the differently treated leaves were incubated at 20° C., theoptimal temperature for M. pinodes spore germination in a day/nightincubator as illumination is necessary for spore germination (Roger &Tivoli, 1996, Mycological Research 100:304-306). After incubation forsix days the foliar lesions were measured and leaf damage compared tothat of the controls.

Example 11 Seed Treatment

Different lots of sorghum seeds were artificially inoculated with eithercovered (Sporisorium sorghi) or loose (Sporisorium cruentum) kernelsmuts spores at the rate of 5% (w/w) before application of seedtreatments. An aerial crude extract of A. africanus was suspended inwater at a rate of 2.0 g/1. Sorghum seed lots of 90 g each were treatedwith 15 ml of the crude extract by mixing thoroughly in a small plasticbag 24 h before planting. A standard synthetic seed dressing fungicide,Thiram (65 W), was applied in the same way at the rate of 0.25% (w/w)per Kg seed and served as a positive control. Sorghum seeds artificiallyinoculated with both loose or covered smuts spores, but were not treatedwith the extract or synthetic fungicide, served as a second control.

Example 12 Field Trial

A field trial was conducted under irrigation at Melkassa ResearchCentre, Ethiopia during 2003. Plots were arranged in a randomisedcomplete block design and treatments were replicated three times.Treated sorghum seeds were planted by hand in five rows, leaving 0.75 cmbetween rows, in 18.75 m² plots. Standard fertilizer was applied andplots were kept at field capacity by means of furrow irrigation. Diseaseincidence was recorded as percentage infected plants. Grain yield wasdetermined on the whole plot.

Example 13 Activity Directed Liquid-Solid Extraction

Dried methanolic crude extracts of the roots (268.5 g) and aerial parts(368.83 g) of A. africanus were fractionated by means of liquid-solidextraction using hexane (DC=2.0), diethyl ether (DC=4.3), ethyl acetate(DC=6.0) and dichloromethane (DC=8.6) as solvents at a ratio of 2 ml/gcrude extract. Extraction was repeated more than 20 times with freshsolvent for each step by shaking vigorously on a mechanical shaker for10 min. The four fractions were collected separately and evaporated todryness under vacuum at 35° C. by means of a Büichi rotavapor. The massof recovered dry material was determined for each fraction. In order toestablish the success of the fractionation process, a thin layerchromatography (TLC) profile was obtained for each fraction with a 0.5mm Silica Gel 60 plate using chloroform:methanol:water (80:20:10) asmobile phase. The mycelial growth inhibitory activity potential of eachsemi-purified extract was subsequently established using F. oxysporum astest organism.

Example 14 Activity Directed Column Chromatography Fractionation

The most active extractants obtained from the liquid solid extractionprocedure were further fractionated using column chromatography. Acolumn (2.6×46 cm) packed with either Sephadex LH20 (Pharmacia) for theroot extracts or Silica gel (0.25; Merck, Darmstadt, Germany) for theaerial parts was employed. The residue of the root (7 g) was elutedsuccessively with ethanol (100%) followed by methanol (100%) and amethanol:water (50:50 v/v) mixture. The column chromatographic residueof the aerial plant parts (5 g) was eluted with a gradient solventsystem of methanol:chloroform (15:85, 20:80, 25:75, 30:70 and 40:60v/v). Elution was adjusted at a flow rate of 3 ml/min.

Approximately 120 ml of the root and aerial part eluent were collected.Those column chromatographic fractions that showed similar Q-TLC profilepatterns were combined separately. Mycelial growth inhibition of F.oxysporum was used to identify active column chromatographic fractionsfor further purification of the active compounds by means of preparativethin layer chromatography.

Example 15 Preparative Thin Layer Chromatography (PTLC)

The most active combined column chromatography fractions were furtherpurified by means of preparative thin layer chromatography (PTLC) usingSilica gel F 1500/LS (1 mm) plates. Fifteen mg of each of the activecolumn fractions were dissolved in 50 μl methanol (100%) and loaded ontothe plate by streaking evenly over the baseline with the aid of a glasscapillary tube. This was repeated 10 times on 10 different plates toseparate compounds from a total of 150 mg of each of the activefractions. The plates were dried in front of a fan between streaking andthen developed in a saturated chamber using a chloroform:methanol:water(80:20:10 v/v) solvent system as mobile phase. Detection of compoundswas done under UV-light at 254 and 365 nm (Wagner and Bladt, 1996, PlantDrug Analysis. A thin layer chromatography atlas. Second edition.Springer, Berlin). Individual compounds were isolated by scraping offthe detected zones of the sorbent layer from the plates using a spatulaand transferred to Eppendorff vials. The compounds were recovered fromthe Silica by elution with methanol (100%), followed by centrifugationfor five minutes at 12000 r.p.m., and tested for antifungal activityafter the methanol was removed by drying at 35° C. in an oven.

Example 16 Qualitative Thin Layer Chromatography (Q-TLC)

Only the most active isolated compounds were again tested for purity inan original analytical thin layer chromatography (TLC) system (Mikes andChalmers, 1979, Laboratory handbook of chromatographic and alliedmethods. Ellis Horwood Ltd., London) using Silica gel 60 F₂₅₄-aluminiumbacked and pre-coated plates. Ten to 15 mg of each sample were loadedonto the plates at the baseline and developed in a saturated chamberusing either chloroform:methanol:water (80:20:10 v/v) ortoluene:acetone:ethyl acetate (7:2:1 v/v; Wagner and Bladt, 1996) assolvent systems. After drying the plates in a stream of air, compoundswere either detected under UV-light at 254 and 365 nm or the plates werestained with 5% (v/v) ethanolic H₂SO₄ or 1% (m/v) Vanillin (1 g in 100ml H₂SO₄; Wagner and Bladt, 1996). Non-pure compounds were againsubjected to preparative TLC acidified with 1% (v/v) HCL until purecompounds were obtained. Only pure compounds that showed the highestantifungal activity were subjected to nuclear magnetic resonance (NMR)spectroscopy in order to identify them and to elucidate their molecularstructures.

Example 17 Nuclear Magnetic Resonance (NMR) Spectroscopy

To identify the most bioactive compounds purified from the roots andaerial plant parts and elucidate their molecular structures, isolatedcompounds were washed repeatedly with acetone to obtain an acceptablelevel of purity. Subsequently, the compounds were submitted to nuclearmagnetic resonance spectroscopy (¹H NMR). NMR-spectroscopy was performedon a Bruker 300 MHz DRX 300 spectrometer at 296K (23° C.) withtetramethylsilane (Si(CH₃)₄; TMS) as the internal standard. The solventsused were deuteriochloroform (CDCl₃), or deuterioactetone [(CD₃)₂ CO] asindicated. Chemical shifts were reported in parts per million (ppm) onthe 6-scale and coupling constants were given in Hz. The followingabbreviations were used: s=singlet, d=doublet, dd=doublet of doublets,m=multiplet, br=broadened, t=triplet. All FAB mass spectra were recordedon a VG 70-70E double-focusing mass spectrometer. Circular dichroism(CD) spectra were recorded on a Jasco J-710 spectropolarimeter withmethanol as solvent. Structural elucidation was achieved viaspectroscopic methods (1D NMR and 2D NMR spectrometry), FAB and EI-MS aswell as chemical methods, such as hydrolysis. Due to the complexity ofthe ¹H NMR spectrum of the non-derivatised sapogenin, a peracetatederivative (2; FIG. 3A) was used in the structural elucidation. This wasachieved via spectroscopic (NMR) and spectrometric (MS) methods, as wellas hydrolysis.

1-38. (canceled)
 39. A preparation suitable for biological plantprotection based on parts of plants from the species Agapanthusafricanus in form of a dry powder, obtainable by the following steps:(i) drying one or more of the different aerial parts of the plant at30-40° C. to the exclusion of sun light; (ii) grinding the dried plantmaterial to a grit size less than 0.1 mm, (iii) soaking the groundmaterial in methanol, thus forming a suspension/solution; (iv)performing a stirred extraction of the suspension; (v) evaporating thesolvent without prior separation of the solid phase from the solubleorganic phase; (vi) soaking the evaporated solid phase residue inethanol and repeating steps (iv) and (v); and (vii) drying theevaporated solid phase residue, thus obtaining a dry powder.
 40. Apreparation according to claim 39, wherein the combined aerial parts areused.
 41. A preparation of claim 39, wherein the flowers are used.
 42. Apreparation according to claim 39, wherein, instead of aerial parts,soil parts of the plant are used.
 43. A preparation according to claim39, further comprising solid, pulverulent carrier materials or fillers,and optionally additives that augment or regulate the effect of thepreparation.
 44. A preparation in form of an aqueous solution orsuspension based on a preparation according to claim
 39. 45. Apreparation of claim 44, wherein the concentration of the dry powder isin the range from 0.25 g/l to 2 g/l.
 46. A plant preparation comprisinga first plant preparation according to claim 39 and at least a secondplant preparation in form of a crude extract, dry powder or an aqueoussuspension or solution thereof, wherein said second plant preparationexerts an additional plant protective effect on the plants or partsthereof treated with the composition, is obtained by analogous processsteps as said first plant preparation, and derives from (i) a speciesfrom the species Tulbaghia violacea, or (ii) a mixture of species of thePink family and Alfalfa species, wherein the proportion by weight of thedried Pink species material is between 80 and 99%.
 47. Use of a plantpreparation according to claim 39 as a biological plant protectiveagent.
 48. Use of a plant preparation according to claim 47, wherein theplant protective agent is an antifungal agent.
 49. Use of a plantpreparation according to claim 48, wherein the antifungal agent inhibitsmycelial growth of fungi.
 50. Use of a plant preparation according toclaim 48, for preventing infection of crop by fungi in vivo under fieldconditions.
 51. Use of claim 50, wherein a plant preparation from thecombined aerial parts of Agaphanthus africanus is used.
 52. Use of claim47, wherein the biological plant protective agent is a bio-stimulatoryagent that induces systemic acquired resistance (SAR) and exerts growthinduction in plants or plant parts treated with the agent.
 53. Use ofclaim 52, wherein the activity or efficacy of said preparation is higherthan the sum of the activities or efficacies of preparations based onthe respective single components of the aerial parts of Agaphanthusafricanus. 54.3-[{O-β-D-glucopyranosyl-(1″-3′)-α-L-rhamnosyl-(1″-2′)}-β-D-glucopyranosyloxy]agapanthegeninor a derivative thereof, wherein all sugar hydroxyl groups areacetylated
 55. A composition suitable for biological plant protectioncomprising a compound of claim 54 and the flavonoid compounds 5,7,4′tri-O-flavanone, 5,7,3′,4′-tetra-O-acetylflavanone andtrans-4,2′,4′-tri-O-acetylchalcone.
 56. Use of the compound of claim 54as antifungal agent that inhibits the mycelial growth of fungi.